Diagnosis of Bacterial Infection Bacterial Cultivation

Outline General procedure of bacteriological Diagnosis Culture medium concept categories bacterial growth patterns Inoculation and transfer techniques

General Procedure of Bacteriological Diagnosis Specimens collection General rules The specimens should be representative of the infectious process; Sufficient material; Avoid the contamination of specimens; Be sent to the lab immediately in an appropriate method and examined ASAP. Be collected before using antimicrobial agents, e.g. antibiotics.

General Procedure of Bacteriological Diagnosis specimens Morphologic identification microscopy & staining Molecular Biological Diagnosis(hybridization, PCR, RT-PCR,etc) Serological diagnosis (Ab titer) convalescent phase / acute phase≥4 Isolation, identification EIA, ELISA, IF test, agglutination test Antigen detection Biochemical tests

Bacterial Cultivation Experiment 1 Bacterial Cultivation

Requirements for bacterial growth Nutrients H2O, C-source, N-source, Inorganic salts, Growth factors Temperature pH Gas incubator  Temperature, gas culture medium  Nutrients, pH

Culture medium Types of Culture Media is the mixture of various nutrients that is suitable for the growth of microorganisms. Types of Culture Media based on the function and chemical components based on the physical state

Based on the function and the chemical components: Basic Medium --contains the basic nutrients for the most bacterial growth; --the base of other kind of media. --e.g. broth. Nutrient Medium/Enriched Medium Additional or special nutrients (e.g., serum, growth factors, trace elements) are added to support some fastidious bacterial growth. e.g. blood agar.

Selective Medium the medium that can prevent the certain bacterial growth while permitting others. e.g. SS agar Differential Medium Some special substrates and indicators are added into the media in order to produce a visual differentiation when several bacteria grow on the same kind of medium. e.g. EMB agar (Eosin-methylene blue agar).

E.coli on EMB S.dysenteriae on EMB

Citrate slant Double sugar iron slant

Anaerobic Medium a medium for the cultivation of certain anaerobes. The medium contains reducing agent, such as non-saturation fatty acid.

Based on the physical state Liquid medium: Without agar. for the proliferation of bacteria. Solid medium: 1.5-2.5% agar. for the isolation and identification of bacteria e.g., slant, Petri dishes/plates. Semisolid medium: 0.3-0.5% agar. for the observation of bacterial motility and preservation of bacteria.

Bacterial growth patterns In liquid medium: Superficial growth; Turbidity/diffuse; Precipitate growing; (sediment) In solid medium: Confluent growth / Smear; Colony: a cluster of microorganisms growing on a solid medium. It is directly visible and arises from a single cell.

In semi-solid medium: Only grow along the line of inoculation Grow diffusely

General procedure of bacteriological Diagnosis Culture medium concept categories bacterial growth patterns Inoculation and transfer techniques

Inoculation and Transfer Techniques Streak-plate technique Slant inoculation Liquid medium inoculation Semisolid medium inoculation

Streak-plate technique four-area streak plate technique I 1/10 II I 1/5 Rotate plate 90 Flame loop Flame loop Rotate 90 III 1/4 IV Rotate 90

Slant inoculation

Liquid medium inoculation

Inoculation method Culture media Bacterial strains 2 students/group Inoculation method Culture media Bacterial strains Streak plate 2 plates (1plate/student) A mixed liquid of S.aureus & E.coli Slant inoculation 2 slants (1slant/student) 1 E. coli slant Liquid medium 1 liquid medium 1 E. coli slant inoculation 1 liquid medium 1 B. subtitis slant Semisolid medium 1 semisolid medium 1 Proteus slant inoculation (Stabbing) 1 semisolid medium 1 S.dysenteriae slant Mark your freshly inoculated plate/tube; Shake up the mixed liquid of bacterial strains before using it. Prepare Gram Stain & Acid-fast Stain. NOTE!!!